Desmosine derivatives and reagent for preparing artificial antigens

ABSTRACT

A novel desmosine derivative, which is useful for preparing a desmosine artificial antigen, has an activated, disulfide bond on the side chain at 3 or 5 position of the pyridinium ring. The derivative can combine with a polymer having thiel groups by a disulfide bond to form the effective artificial antigen.

This is a division, of application Ser. No. 839,576, filed 3/14/86 , nowU.S. Pat. No. 4,723,012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel desmosine derivatives and theirapplication.

2. Description of the Prior Art

In the lungs, a balance is preserved between the elastase, one of theproteases, and antielastase. Namely, as can be inferred the network ofthe elastic fibers of the lungs is protected by the action of theantielastase from the damages arising from its extension. When thebalance is destroyed due to some cause, the elastin (elastic fibers),the main component of the lungs, undergoes an unlimited decomposition,resulting in the destruction of pulmonary cells and consequently chronicobstruction pulmonary disease (COPD). If a highly sensitive chemicalanalytic process is available which can determine the manner in whichelastin is decomposed in the living organism, it will be possible toeffectively detect changes in the morbid condition of various tissuesand organs including the lungs as early as possible. When decomposed,elastin is excreted into the urine in the form of peptide containingcross-linded amino acid such as unique desmosine found in elastin alone.Consequently analysis of a quantity of desmosine excreted into the urinewill ensure the discovery of the occurrence of pulmonary diseases.

Recently Harel et al, Am. Rev. Respir. Dis., Vol 122, p 769, 1980, andKing et al Connect. Tissue Res., Vol 7, P 263, 1980 disclosed theprocess of analyzing desmosine contained in the urine by means ofradioimmunoassay (RIA). However, since RIA method involves the use ofradioisotopes, it presents various difficulties such as not only hazardsto the human body, but also environmental pollution, difficulties inwaste disposed, establishment of an extra facility required for theapplication of radioisotopes, and problems concerning the applicationperiod of radioisotopes due to their half-life. Consequently the RIAmethod involves numerous difficulties in being accepted as a generalanalytic method and is obstructed in rapid dissemination. Moreover, theartificial desmosine antigen developed by Harel et al or King et al isprepared by combining desmosine and protein at random using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Therefore,the artificial desmosine antigen proposed by the above-mentionedresearchers gives an antibody having only an extremely low titer. Theiranalytic method only has the maximum merit of diluting antiserum to theextent of 200 to 250 times magnification. In this respect, too, the RIAmethod presents great difficulties in wide dissemination.

In view of the above-mentioned difficulties, it has become necessary todevelop a new desmosine-analyzing method based on the enzymeimmunoassay(EIA). The EIA offers the advantages that less hazards are presented tothe human body than RIA process when a clinical chemical examination isperformed; difficulties are eliminated in dumping waste leading toenvironmental pollution; it is unnecessary to provide any extra facilityfor its application; reagents involved can be safely stored over a longperiod time; and wide dissemination is ensured.

Nevertheless, the EIA process is generally considered to have such anunsatisfacfory detection sensitivity as is ten times lower than the RIAprocess. Therefore, antiserum applied to the EIA process is demanded tohave an extremely high antibody titer.

SUMMARY OF THE INVENTION

It is accordingly an object of this invention to provide a novelsubstance which can be applied to the preparation of an artificialantigen which ensures the derivation of antidesmosine antiserum havingan extremely high antibody titer value.

To attain the above-mentioned object, the present invention provides adesmosine derivative represented by the following formula: ##STR1##where R is an electron-attracting organic group, X.sup.⊖ is an anion,and n is an integer of 1 to 6.

Further objects of the present invention is to provide a reagent forpreparation of an artificial antigen intended for the production ofantidesmosine antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended FIGURE is a graph showing the result of theenzymeimmunoassay of desmosine by applying the antidesmosine antiserumderived from an artificial antigen bonding a desmosine derivativeaccording to this invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compound of this invention is a novel substance containing, as seenfrom Formula 1, a disulfide group in the side chain at 3 or 5 positionof the pyridinium ring. In Formula I, R represents anelectron-attracting organic group. Preferably, R is an aromatic group.Examples of the group R include pyridyl, azidophenyl, and phenyl.

In Formula 1, X.sup.⊖ denotes proper anions such as acetate, formate,chlorine, bromine and iodine ions. When, however, a desmosine derivativeof this invention is applied as a reagent for the preparation of anartificial antigen to derive an antidesmosine antigen, a portion actingas an antigenic determinant is the cation portion, and the anion X.sup.⊖makes no contribution to an immune response reaction. Therefore, theanion X.sup.⊖ need not be limited to the foregoing examples. In FormulaI, n denotes an integer of 1 to 6, and preferably 2 to 4.

The desmosine derivative of this invention can be prepared by reactingdesmosine or its salt with activated carboxylic acid represented by theformula:

    A-OC--CH.sub.2).sub.n S-S-R                                (II)

where R and n are the same as in Formula I, and A is ethoxy group,##STR2##

Examples of the activated carboxylic acid of Formula II are

ethoxycarbonyl-3-(2-pyridyldithio)propionate,

ethoxycarbonyl-4-(2-pyridyldithio)butytate,

ethoxycarbonyl-5-(2-pyridyldithio)valerate,

ethoxycarbonyl-6-(2-pyridyldithio)capronate,

ethoxycarbonyl-7-(2-pyridyldithio)caprylate,

N-succinimidyl-3-(2-pyridyldithio)propionate,

N-succinimidyl-4-(2-pyridyldithio)butyrate,

N-succinimidyl-5-(2-pyridyldithio)valerate,

N-succinimidyl-6-(2-pyridyldithio)capronate,

N-succinimidyl-7-(2-pyridyldithio)caprylate,

N-maleinimidyl-3-(2-pyridyldithio)propionate,

N-maleinimidyl-4-(2-pyridyldithio)butyrate,

N-maleinimidyl-5-(2-pyridyldithio)valerate,

N-maleinimidyl-6-(2-pyridyldithio)capronate,

N-maleinimidyl-7-(2-pyridyldithio)caprylate,

ethoxycarbonyl-3-(4-azidophenyldithio)propionate,

ethoxycarbonyl-4-(4-azidophenyldithio)butyrate,

ethoxycarbonyl-5-(4-azidophenyldithio)valerate,

ethoxycarbonyl-6-(4-azidophenyldithio)capronate,

ethoxycarbonyl-7-(4-azidophenyldithio)caprylate,

N-succinimidyl-3-(4-azidophenyldithio)propionate,

N-succinimidyl-4-(4-azidophenyldithio)butyrate,

N-succinimidyl-5-(4-azidophenyldithio)valerate,

N-succinimidyl-6-(4-azidophenyldithio)capronate,

N-succinimidyl-7-(4-azidophenyldithio)caprylate,

N-maleinimidyl-3-(4-azidophenyldithio)propionate,

N-maleinimidyl-4-(4-azidophenyldithio)butyrate,

N-maleinimidyl-5-(4-azidophenyldithio)valerate,

N-maleinimidyl-6-(4-azidophenyldithio)capronate and

N-maleinimidyl-7-(4-azidophenyldithio)caprylate.

According to the present invention, the reaction of desmosine with theactivated carboxylic acid is carried out at 0° to 30° C. preferably 15°to 20° C., and for 1 to 5 hours. The desmosine and the activated acidare used in a molar ratio of 1:0.5 to 1:1.5, preferably 1:1.05.

It is important that the above-mentioned reaction be performed in aphosphate buffered solution with the pH value of 5.0 to 6.5 Reactionunder this pH condition causes the activated carboxylic acid toselectively react with the amino group on the side chain at 3 or 5position of the pyridinium ring of desmosine, thereby forming an amidebond. The reaction product can be isolated by means of columnchromatography as described in Example 1.

The desmosine derivative of this invention is bonded with a carrierpolymer having a thiol group to prepare an antigen. Reaction between thedesmosine derivative and carrier polymer is thiol-disulphide exchangereaction. This reaction causes the desmosine portion of the desmosinederivative to be bonded with the polymer carrier by the disulfide bond,with the elimination of R-SH. This process provides an artificialantigen for derivation of antidesmosine antibody. The thiol-disulfideexchange reaction is carried out at a temperature of 0° to 30° C. for aperiod of 1 to 5 hours, with the desmosine derivative and the carrierpolymer being used in a molar ratio of 10:1 to 20:1.

The carrier polymer applied in the preparation of an artificial antigenmay be natural or synthetic, provided the molecule of the polymercontains a thiol radical and has a biocompatibility. The carrier polymerincludes peptide (including protein) and other polymers. Specificexamples of the carrier polymer are those derived from bovine serumalbumin (BSA), mouse gamma globulin (MGG), mouse serum albumin (MSA),and keyhole limpet hemocyanin by reducing their intramolecular disulfidebonds with dithiothreitol to thiols.

As seen from the formula (I), one molecule of the desmosine derivativeof the invention is bonded with the carrier polymer by means of onebridge linkage without any change of the desmosine structure,particularly the other three amino acid residual groups bonded with thepyridinium ring. As a result, it is possible to provide an artificialantigen having an antigenic determinant structure fully exposed to themolecule surface. Therefore, this artificial antigen ensures thepreparation of antidesmosine antiserum having an extremely high antibodytiter.

EXAMPLE 1

Preparation of4-(4-amino-4-carboxybutyl)1-(5-amino-5-carboxypentyl)-5-(3-amino-3-carboxypropyl)-3-[3-carboxy-3-{3-(2-pyridildithio)propionyl}amino]propylpyridiniumacetate.

Desmosine, 39 mg, was dissolved in phosphate buffered solution (0.05M,pH 5.9) containing 25 ml of 0.1M NaCl in a triangular flask of 50 ml.Separately, 23 mg of N-succinimidyl-3-(2-pyridyldithio)propionate wasdissolved in 3 ml of ethanol. The latter solution was slowly dripped inthe desmosine solution. The reaction mixture was stirred for 3 hours atroom temperature. After reaction, the solvent was removed by means of arotary evaporator. The residue was poured into a column (1.5 cm indiameter and 90 cm in height) of Sephadex G-25 Super Fine available fromPharmacia Fine Chemicals Co., and eluted with an acetate bufferedsolution (0.1M, pH 4.5) at the flow rate of 0.1 ml/min. The eluate wascollected in test tubes at the rate of 3 ml. First, unreacted desmosine(the 26th to 29th test tubes) was eluted. Then the desmosine derivativeof this invention (the 33rd to 35th test tubes) was eluted. Last, theby-products (the 37th to 40th test tubes) were eluted. The solvent wasremoved from the fractions containing the desmosine derivative of thisinvention by means of a rotary evaporator. The residue was poured into acolumn (2.5 cm in diameter and 91 cm in height) of Biogel P-2-(-400mesh) available from Rio Rad Laboratories Co., and eluted with 0.1M ofacetic acid to effect separation and desaltation. After freeze-dried,15.6 mg of light yellow solid was obtained (yield 30%). This lightyellow solid was analyzed by means of ¹ H NMR, mass spectrum (MS) andultraviolet absorption spectrum (UV). The product proved to be thedesired desmosine derivative having a purity of 97% or more. Theanalytical data obtained were as follows:

¹ H NMR (solvent D₂ O, internal standard DSS) (ppm): 1.40 (m, 1H), 1.47(m, 1H), 1.59 (m, 1H), 1.67 (m, 1H), 1.89 (m, 2H), 2.06 (s, 3H),1.96-2.11 (m, 4H), 2.17 (m, 4H), 2.78 (m, 2H), 2.90 (m, 4H), 3.02 (m,2H), 3.12 (t, 2H), 3.73 (t, 1H), 3.79 (m, 1H), 3.88 (m, 2H), 4.48 (t,2H), 7.30 (t, 1H), 7.84 (m, 2H), 8.39 (d, 1H), 8.46 (s, 1H), 8.52 (s,1H).

MS (m/z): 723

UV (solvent 0.1N acetic acid): λ (max); 236 nm, 276 nm (shoulder, 270nm)

EXAMPLE 2

Preparation of4-(4-amino-4-carboxybutyl)1-(5-amino-5-carboxypentyl)-5-(3-amino-3-carboxypropyl)-3-[3-carboxy-3-{3-(4-azidophenyldithio)propionyl}amino]propylpyridiniumacetate.

Desmosine tetraacetate, 38 mg (65 μmol), was dissolved in phosphatebuffered solution (0.05M, pH 5.9) containing 25 ml of 0.1M NaCl in atriangular flask of 50 ml. Separately, 21 mg ofN-succinimidyl-3-(4-azidophenyldithio)propionate was dissolved in 3 mlof dimethyformamide. The latter solution was slowly dripped in thedesmosine solution. The reaction mixture was stirred for 3 hours at roomtemperature. After reaction, the solvent was removed by means of arotary evaporator. The residue was poured into a column (2.5 cm indiameter and 91 cm in height) of Biogel P-2-(-400 mesh), and eluted with0.1M of acetic acid. The eluate was collected in a volume of 10 ml intest tubes. The fractions collected in the 23rd to 25th test tubes werefreeze-dried to obtain 25 mg of the desired product as a light yellowsolid.

EXAMPLE 3

Preparation of an artificial antigen

The intramolecular disulfide bonds of bovine gamma-globulin (BGG) wasreduced by dithiothreitol to be converted into thiols according to themethod described in Analytical Biochemistry, 94, 253-258 (1979). Thethiolated BGG, 20 mg, was dissolved in 10 ml of a phosphate bufferedsolution (pH 7.5, 0.1M) containing 0.1M NaCl and 6M urea. Desmosinederivative, 4.67 mg, obtained in Example 1 was added. The mixture wasstirred at room temperature for 45 minutes. 15 ml of 10% trichloroaceticacid was added to the reacted solution. The mixture was subjected tocentrifugal separation at a rotation rate of 3000 rpm for 10 minutes.The supernatant was taken off. 1 ml of water was added to the whiteprecipitate and washing was carried out by centrifuge. The whiteprecipitate was freeze-dried, producing 17 mg of the desired antigen aswhite powder. The bonded molar ratio between the desmosine portion ofthe antigen and BGG was calculated to be 10 by the process ofdetermining the ultraviolet ray absorbance at 343 nm of theabove-mentioned reacted solution to quantitatively analyzepyridine-2-thione released by the thiol-disulfide exchange reaction,with the molecular weight of thiolated BGG assumed to be 150,000.

EXAMPLE 4

Derivation of antidesmosine antiserum

0.3 ml of 8M urea was dissolved in 1.45 mg of the antigen obtained inExample 3. 0.5 ml of physiological saline (0.9%) was added. The mixturewas thoroughly mixed with the equivalent amount of Freund's completeadjuvant for emulsification. The preparation was injected into the sixportions of the inner surfaces of the toes of each of three white maleNew Zealand rabbit weighing 3.5 kg. Nine days later, a secondimmunization was undertaken by the process of thoroughly dissolving 0.1ml of 8M urea in 1.53 mg of desmosine artificial antigen, adding 0.6 mlof physiological salt solution (0.9%), thoroughly mixing the mixturewith the equivalent amount of Freund's incomplete adjuvant foremulsification, and subcutaneously injecting the preparation into 10portions of the rabbit's back. Later, five immunization steps were takenevery two weeks by injecting a preparation obtained by emulsifying 0.1mg of the desmosine derivative in 100 microliters of physiologicalsaline into the ear vein of the rabbit. All three immunized rabbitsshowed an increase in the antibody titer, and a dose-response curvecould be drawn. Thus, it was proved that the antibody obtained from theartificial antigen prepared from the desmosine derivative of thisinvention was specific to desmosine. On the 11th day after the finalinjection (91 days after the initial immunization), the total bloodcollection was undertaken through the carotid artery of the rabbit whichhad been subjected to starvation from the day preceding the bloodcollection. Thus, the antidesmosine antiserum was obtained.

EXAMPLE 5

EIA for desmosine

Desmosine was enzyme-immunoassayed using the antidesmosine antigenobtained in Example 4.

A predetermined quantity of a complex of desmosine with keyhole limpethemocyanin, obtained by the carbodiimide method through the customarystep, was coated in the wells of an EIA microplate of the 96 well type,thereby bonding the desmosine to the wells. The wells were further addedwith solutions of various concentrations of free desmosine to be used asanalytic samples. Then an antigen-antibody reaction was carried out byprogressing filling the wells with the antiserum in such a manner thatthe antiserum held in the 96th well was finally diluted to 30,000 timesthe original quantity. After reaction, the free desomosine wasseparated, and the antidesmosine antibody reacted with the bondeddesmosine and alkaliphosphatase-labeled goat (antirabbit IgG) antibodywere bonded together. Measurement was made of the enzyme activity of thebonded mass. The above-mentioned method determines the concentration ofthe free desmosine by measuring the competitive inhibition by the freedesmosine against the antigen-antibody reaction between theantidesmosine antigen and the bonded desmosine. As seen from theappended chart, therefore, the application of the 30,000 times dilutedantiserum ensured the determination of the minute amount (10⁻⁹ g/ml) ofdesmosine. The percentage inhibition against the reaction between thebonded desmosine and antidesmosine antigen can be determined from theformula:

    Percentage inhibition (%)=(A-B)/A)×100

where A is enzyme activity of a desmosine-free sample (control), and Bis enzyme activity of a desmosine-containing sample.

The results of the foregoing experiments prove that the antidesmosineantiserum derived from the artificial antigen bonded with the desmosinederivative of this invention can be satisfactorily applied in analysiseven though the antidesmosine antiserum may be diluted over 100 timesmore than the conventional antiserum, namely, has an extremely highantibody titer.

What is claimed is:
 1. A method for preparing a desmosine artificialantigen comprising the step of reacting a desmosine derivative having aformula: ##STR3## where R is an electron-attracting organic groupselected from the group consisting of pyridyl, azidophenyl and phenyl,X⁻ is an anion, and n is an integer of 1 to 6, with a carrier polymerhaving a thiol group derived from a material selected from the groupconsisting of bovine serum albumin, mouse gamma globulin, mouse serumalbumin and keyhole limpet hemocyanin with a molar ratio of thedesmosine derivative to carrier polymer being from about 10:1 to about20:1 at a temperature and for a period of time sufficient for bondingsaid desmosine derivative to said carrier polymer through a disulfidebond, with the elimination of R-SH, to produce a desmosine artificialantigen.
 2. The method according to claim 1, wherein said reaction isconducted at a temperature of from about 0° to 30° C.
 3. The methodaccording to claim 1, wherein said reaction is conducted for about 1 toabout 5 hours.
 4. The method according to claim 1, wherein R is2-pyridyl, and n is
 2. 5. The method according to claim 1, wherein R isazidophenyl and n is 2.